Retinal photoreceptor cells maintain specific composition of the photosensitive membranes by continuous membrane renewal. In patients with retinal genetic disorders this process is often compromised by mutations in rhodopsin or factors that regulate its intracellular trafficking. For example the C-terminal domain of rhodopsin is a frequent target for mutations that cause severe forms of autosomal dominant retinitis pigmentosa (ADRP) while mutations in regulators of small GTPases of the ras and rab super family cause choroideremia and X- linked RP3. The long-term goal is to delineate the molecular mechanisms that regulate polarized delivery of rhodopsin and its associated proteins to the rod outer segment. Based on our preliminary studies, we propose to investigate interactions between rhodopsin, small GTPases and their regulatory proteins and phospholipids that in normal cells bring about the correct membrane sorting and delivery to the ROS. We will apply biochemical, morphological and cell biological tools to analyze photoreceptor biosynthetic membranes under normal conditions and under normal conditions that correspond to those found in retinal diseases. The specific aims of this proposal are: 1) To determine the role of rhodopsin in regulation of its post-Golgi trafficking. 2) To elucidate the role of small GTPases, their regulators such as Retinitis Pigmentosa GTPase Regulator (Rpgr), and post-Golgi membrane specific proteins in rhodopsin trafficking. 3) To elucidate the role of lipids in the polarized membrane delivery to the ROS. 4) To determine the mechanism of global regulation of membrane delivery to the ROS by extracellular signals and secondary messengers. Rhodopsin trafficking will be monitored in vivo and in an established cell-free system that reconstitutes sorting of rhodopsin into specific post-Golgi carrier membranes. Biochemical analyses of retinal subcellular factions will be complemented by confocal and electron microscopy. To model conditions found in retinal diseases membrane renewal will be perturbed by adding antibodies, inhibitors, peptides and mutant proteins, or by depleting proteins or lipids that participate in intracellular sorting and trafficking. Our research goals are to provide better understanding of the molecular mechanisms that govern cell polarity and the health of retinal photoreceptors. With this knowledge we will be able to design and develop new approaches to the treatments of retinal diseases.